1. Field of Invention
This invention relates to a process for the fractionation of polymers that are aqueous soluble, and do not contain functional groups capable of carrying a charge at neutral pH.
2. Description of Prior Art
Many methods have been described for separating polymers of similar composition and structure. See for example U.S. Pat. Nos. 5,028,336, 5,116,508, 5,523,492, 5,567,859, 5,696,298, 5,800,711 and European Patent Application WO 92/16484, incorporated herein by reference. Also, various procedures have been described to fractionate proteins and peptides, but most include precipitation using ammonium sulfate (Englard and Seifter, 1990). This method relies on the fact that proteins, in an aqueous solution, maintain a tertiary structure based on their amino acid composition and various bonds within the molecule. The tertiary structure generally allows the hydrophobic substituents to be sequestered inside the molecule and the hydrophylic components to be on the surface, and in contact with the aqueous environment. Changes in the ionic strength of the aqueous solution cause unfolding of the molecule, and with exposure of the hydrophobic substituents to the aqueous environment, the solubility of the protein decreases and h precipitates. By carefully adjusting pH, ionic strength, and sometimes temperature, it is frequently possible to separate proteins with similar amino acid sequences (Englard and Seifter, 1990, King, 1972, Tarli and Li, 1974). Clearly, this method is useful only for proteins and other polymers composed of substituents that vary considerably in polarity, and therefore aqueous solubility. This is not the case for many synthetic polymers.
A method to separate water soluble organic electrolytes in an aqueous medium from other water soluble hydrocarbons has been disclosed (Bartels and Reale, 5,028,336). The pH of the aqueous medium is adjusted so that most of the organic electrolytes are charged. The aqueous medium is then passed through a filtration membrane which carries the same charge. The organic electrolytes are repelled by the charge on the membrane, and therefore do not cross. Water and uncharged organic molecules pass through the membrane and are thus separated from the organic electrolytes. This method is limited to organic molecules such as carboxylic acids which contain functional groups capable of carrying a charge at some pH.
Methods relying on supercritical fluid extraction have been developed to separate high molecular weight compounds, including polymers, from complex mixtures in aqueous solution (See for example Kumar and Hedrick, 5,116,508). This method requires a mobile phase of highly compressed gas, such as CO2, at or above its critical temperature and pressure, to be pumped through the aqueous solution. The composition of the mobile phase can be modified to enhance extraction of the desired analyte. Such modifications include using a mixture of gases as the mobile phase, or adding a modifying chemical to the supercritical fluid. Such methods can be conducted on a commercial scale, and used to separate uncharged polymers including polyols. Nevertheless, to be effective, the compressed gases often must be maintained at high temperatures and pressures, which requires complex, well controlled equipment. This frequently makes supercritical fluid extraction an expensive process and limits its commercial applications.
Synthetic polyols such as poly(ethylene glycol) and polyoxyalkylene block copolymers have been used in various medical and pharmaceutical applications including treatment of sickle cell disease, reduction of blood viscosity, treatment of tissue ischemia, treatment of tissue following electrical injury, and drug delivery (Emanuele 5,691,387, Lee, R. C., 5,605,687, Reeve, L. E., 1997). These linear polymers are generally synthesized by repeated sequential reactions that add monomeric subunits to each end of the polymeric chain. Since subunits may add to either or both ends of individual chains at variable rates, the end product is a mixture of molecules varying in molecular weight.
The poly(ethylene glycol)s are composed entirely of ethylene oxide residues linked by ether linkages and vary considerably in molecular weight. These synthetic polymers have been used extensively in drug delivery to solubilize pharmaceutically active compounds. Recently, they have been used to derivatize proteins, peptides and small molecules to prolong half-life and enhance delivery within the body ( ). They have also been derivatized and used as cross-linking components in medical devices. For optimal safety and efficacy in medical applications, these recent uses require polymers of uniform molecular weight having minimal contamination with reaction byproducts.
The poloxamers are polyoxyalkylene block copolymers composed of two polyexyethylene blocks separated by a polyoxypropylene center block. The commercially available product contains a mixture of polyoxyethylene homopolymer, and polyoxyethylene/polyoxypropylene diblock polymers in addition to poloxamer molecules of varying molecular weights. These factors cause the polymer product to have a broad molecular weight range, reflected in a high polydispersity index. The mono- and diblock polymers are generally of a lower molecular weight than the average for the polymer product and contain some unsaturation. When commercially available poloxamers (purchased from BASF Corp.) were analyzed by gel permeation chromatography, a bimodal molecular weight distribution was observed (Reeve, L. E., 1997). The mono- and diblock contaminants, including the unsaturated species, partitioned into the lower molecular weight fraction.
European Patent Application WO 92/16484 discloses the use of gel permeation chromatography to isolate a fraction of poloxamer 188 that exhibits beneficial biological effects, without causing potentially deleterious side effects. The copolymer thus obtained had a polydispersity of 1.07 or less, and was substantially saturated. The potentially harmful side effects were shown to be associated with the low molecular weight, unsaturated portion of the polymer, while the medically beneficial effects resided in the uniform higher molecular weight material. Other similarly improved copolymers were obtained by purifying either the polyoxypropylene center block during synthesis of the copolymer, or the copolymer product itself (Emanuele 5,523,492, Emanuele 5,696,298). Although an effective means of purification, gel permeation chromatography is impractical for the preparation of large quantities of the fractionated polyoxyalkylene block copolymer.
A super critical fluid extraction technique has been used to fractionate a polyoxyalkylene block copolymer as disclosed in U.S. Pat. No. 5,567,859. A purified fraction was obtained, which was composed of a fairly uniform polyoxyalkylene block copolymer having a polydispersity of less than 1.17. According to this method, the lower molecular weight fraction was removed in a stream of CO2 maintained at a pressure of 2200 pounds per square inch (psi) and a temperature of 40xc2x0 C. As is frequently the case, this super critical fluid extraction method required equipment that can control temperature and accommodate compressed CO2 at high pressure. Clearly, these requirements add expense to the procedure and limit its commercial value.
U.S. Pat. No. 5,800,711 discloses a process for the fractionation of polyoxyalkylene block copolymers by the batchwise removal of low molecular weight species using a salt extraction and liquid phase separation technique. Poloxamer 407 and poloxamer 188 were fractionated by this method. In each case, a copolymer fraction was obtained which had a higher average molecular weight and a lower polydispersity index as compared to the starting material. However, the changes in polydispersity index were modest and analysis by gel permeation chromatography indicated that some low molecular weight material remained. The viscosity of aqueous solutions of the fractionated polymers was significantly greater than the viscosity of the commercially available polymers at temperatures between 10xc2x0 C. and 37xc2x0C., an important property for some medical and drug delivery applications. Nevertheless, some of the low molecular weight contaminants of these polymers are thought to cause deleterious side effects when used inside the body, making it especially important that they be removed in the fractionation process. As a consequence, polyoxyalkylene block copolymers fractionated by this process are not appropriate for some medical uses.
Aqueous two phase systems have been used extensively to concentrate or isolate polymers, other large molecules, and even particles from complex mixtures (Hatti-Kaul, R., 2000). Such systems generally avoid the use of organic solvents, and extremes of pH or temperature, and, because of their mild conditions, have been shown to be especially useful to isolate amino acids, peptides, proteins, plasma membranes including membrane vesicles, and viruses. These systems are composed of either hydrophilic polymer pairs or a polymer and a salt that are incompatible in aqueous solution and form two phases in equilibrium with each other. Separations can be carried out using either batch procedures or counter-current distribution. Although widely used for the isolation and purification of biomaterials, aqueous two-phase systems have been used much less extensively for the isolation or fractionation of synthetic polymers.
The present invention is a method for the fractionation of water-soluble synthetic polymers by physically separating the lower molecular weight polymer molecules, byproducts, and contaminants from the desired polymer by using an aqueous two-phase system.
The present invention provides a process for the fractionation of aqueous soluble polymers comprising dissolving a known amount of the polymer in water, adding a soluble extraction salt to the polymer solution, maintaining the solution at a constant optimal temperature for a period of time adequate for two distinct phases to appear, and physically separating the phases. Additionally, the phase containing the polymer fraction of the preferred molecular weight may be diluted to the original volume with water, extraction salt added to achieve the original concentration, and the separation process repeated as needed until a polymer having a narrower molecular weight distribution than the starting material and optimal physical characteristics can be recovered.
It is an object of the present invention to provide a method that separates lower molecular weight polyol polymer molecules from those of very similar chemical composition but higher molecular weight thereby narrowing the molecular weight distribution.
It is another object of the present invention to provide a method to remove reaction byproducts from the polyol polymer.
Another object of the present invention is to provide an improved method which produces a polyol polymer with improved chemical and physical characteristics including decreased polydispersity, decreased unsaturation, higher viscosity in aqueous solution, and hence increased gel strength, and a well defined transition temperature for those polyols that form gels in solution.